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Introduction: The main goal in transplantation is to induce specific rather than general immunosuppression. The CD45 protein is an essential regulator of T and B cell antigen receptor signaling. We have previously shown that T cells expressing low levels of CD45RC have strong immunoregulatory properties while T cells expressing high levels of CD45RC are essentially effector cells. Thus, we investigated the effect of depleting CD45RC+ cells on graft survival.

Methods: Tregs were analyzed for the expression of CD45 isoforms by flow cytometry. LEW-1A rats were grafted in a heterotopic position with a LEW-1 W heart. NSG mice were 2Gy-irradiated or grafted with human skin and injected with 5x106 or 1,5x107 human PBMCs respectively. Animals were treated with 0.8 mg/kg/2.5d for 20d of anti-CD45RC mAb (OX22 or MT2 clones) or isotype. Humoral responses were assessed after KLH + CFA or exogenous red blood cells immunization. FACS Aria-sorted Tregs were i.v. transferred in 4,5Gy-irradiated recipients. CD45RClow/- Tregs were analyzed by RNA sequencing.

Results: First, we have shown that CD45RC is the only CD45-isoform not expressed by Foxp3+ Tregs both in human and rat. Interestingly, transient treatment with anti-CD45RC Mabs induced transplant tolerance in a fully mismatch cardiac allograft model in rat. Importantly, this treatment prevented specifically anti-donor humoral responses while preserved primary and memory responses against cognate antigens. As expected, anti-CD45RC Mabs induced rapid cell death of CD45RC+ T cells through intrinsic cell signaling while preserved CD45RClow/- Tregs. Furthermore, the short-term treatment induced a transient homeostatic proliferation and sustained transcriptional changes in long-term CD8+ and CD4+ CD45RClow/- Tregs that maintained an indefinite, dominant and donor-specific tolerance, as adoptive transfer of Tregs succeeded in this allograft combination only. Finally, we translated these findings to human, in humanized mice models of xenogeneic GVHD and human skin allograft rejection. We demonstrated that ex vivo depletion of CD45RC+ PBMCs prevented development of GVHD, and that transient treatment with anti-human CD45RC Mabs delayed GVHD development and allograft rejection.

Conclusion: This is the first proof of concept that targeting CD45RC with short-term Mabs treatment is an efficient innovative strategy to induce a donor-specific tolerance in transplantation without any other treatment.